Hepatitis C virus (HCV) infects approximately 4 million persons in the U.S. and is a major cause of acute and chronic liver disease, often leading to cirrhosis and hepatocellular carcinoma. The viral factors that are responsible for replication of the HCV genome are poorly understood, and detailed biochemical characterization of these factors is needed to aid in rational development of anti-HCV therapies. The HCV RNA helicase, NS3, is believed to be essential for viral replication. Currently, no rigorous, biochemical models for RNA helicase activity exist, making this class of enzymes one of the least understood viral factors. The goal of this research proposal is to fully characterize the biochemical mechanism of NS3 including its interaction with substrates and other HCV proteins. Genetic methods using the yeast two and three hybrid systems are being applied to identify novel interactions among HCV proteins as well as with specific regions of HCV RNA. Biochemical methods are being used to determine the specificity and thermodynamic parameters that govern such interactions. RNA unwinding by NS3 is being studied using a new pre-steady-state assay in which the enzyme is assembled in a stoichiometric fashion on well-defined oligonucleotide substrates. Using this assay, we will determine whether NS3 functions through a stoichiometric or catalytic mechanism by measuring the active form of the enzyme. A second new assay will be applied to determine whether there exists a directional bias in translocation of NS3 on single-stranded nucleic acid substrates. The results from these experiments will provide the frame work in which to develop a minimal kinetic mechanism of NS3 which is necessary for quantitative understanding of the function of this enzyme. We will test several proposed mechanisms for translocation and unwinding using a combination of nucleic acid footprinting and protein-DNA crosslinking. The specific role of key amino acids in the function of NS3 will also be addressed using site-directed mutagenesis and x-ray crystallography. Preliminary data suggests that NS4b protein interacts with NS4a, which is known to form a tight complex with NS3. Thus, the influence of NS4b on the activity of NS3 will be determined. Results from this project will identify and characterize novel, specific interactions among NS3, its substrates, and other HCV proteins in a quantitative manner. This work will provide the initial step toward our long term goal of recapitulating HCV replication in vitro using biologically relevant proteins and RNA.